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Visual Detection of Rf2 Event in Transgenic Rapeseed (Brassica napus L.) Using Loop-Mediated Isothermal Amplification

Received: 19 October 2022     Accepted: 2 November 2022     Published: 29 November 2022
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Abstract

Specific methods have been developed in order to efficiently detect GMOs (genetically modified organisms) in both feed and food. Current approaches often rely on thermal cycling devices such as PCR amplifier, which makes it difficult for applications in the wild. Therefore a visual LAMP (Loop-Mediated Isothermal Amplification) method for rapid screening Rf2 event in transgenic rapeseed (Brassica napus L.) is established in this paper. For primer design, position 167-366 in the right border junction sequence of B. napus trangenic line Rf2 (accession number: EU090197.1) was chosen as the amplicon. Reaction mixture (at the volume of 25 μL) consisted of 12.5 μL 2×buffer mix, 1.28 μM FIP, 1.28 μM BIP, 0.16 μM F3, 0.16 μM B3, 480 U/mL Bst DNA polymerase, 2.0 μL DNA template and 5.0 μL ddH2O. Dye (SYBR Green I) was pre-added onto the tube lid. The reaction tube was incubated at 60°C for 60 min, followed by heating at 80°C for 10 min to end it. To mix dye with reaction mixture, the tube was centrifuged for 60 s at 8,000 r/min. Corresponding analysis results indicate that this LAMP assay is highly specific and sensitive (1.15×103 copies/μL). In one word, the visual LAMP method specific for Rf2 event in rapeseed, which turns out to be simple, time-effective, sensitive without relying on expensive instruments, is suitable for quick screening in ports.

Published in Journal of Plant Sciences (Volume 10, Issue 6)
DOI 10.11648/j.jps.20221006.14
Page(s) 222-226
Creative Commons

This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.

Copyright

Copyright © The Author(s), 2022. Published by Science Publishing Group

Keywords

Brassica Napus, Rf2 Event, LAMP, Quick Screening, Ports

References
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[3] Radchuk VV, Klocke E, Radchuk RI, et al. Production of transgenic rape plants (Brassica napus L.) using Agrobacterium tumefaciens. Genetika, 2000, 36 (7): 932-941.
[4] Pua EC, Mehra-Palta A, Nagy F, et al. Transgenic Plants of Brassica napus L. Nature Biotechnology, 1987, 5: 815-817.
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[14] Gao H, Wen L, Tian J, et al. A portable electrochemical immunosensor for highly sensitive point-of-care testing of genetically modified crops. Biosensors and Bioelectronics, 2019, 142: https://doi.org/10.1016/j.bios.2019.111504.
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[18] Sohn SI, Pandian S, Oh YJ, et al. A Review of the Unintentional Release of Feral Genetically Modified Rapeseed into the Environment. Biology, 2021, 10 (12): https://doi.org/10.3390/biology10121264.
[19] Becherer L, Borst N, Bakheit M, et al. Loop-mediated isothermal amplification (LAMP)–review and classification of methods for sequence-specific detection. Analytical Methods, 2020, 12 (6): 717-746.
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Cite This Article
  • APA Style

    Wang Jiaying, Zhao Lei, Li Wen, Huang Suwen, Zhang Jihong. (2022). Visual Detection of Rf2 Event in Transgenic Rapeseed (Brassica napus L.) Using Loop-Mediated Isothermal Amplification. Journal of Plant Sciences, 10(6), 222-226. https://doi.org/10.11648/j.jps.20221006.14

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    ACS Style

    Wang Jiaying; Zhao Lei; Li Wen; Huang Suwen; Zhang Jihong. Visual Detection of Rf2 Event in Transgenic Rapeseed (Brassica napus L.) Using Loop-Mediated Isothermal Amplification. J. Plant Sci. 2022, 10(6), 222-226. doi: 10.11648/j.jps.20221006.14

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    AMA Style

    Wang Jiaying, Zhao Lei, Li Wen, Huang Suwen, Zhang Jihong. Visual Detection of Rf2 Event in Transgenic Rapeseed (Brassica napus L.) Using Loop-Mediated Isothermal Amplification. J Plant Sci. 2022;10(6):222-226. doi: 10.11648/j.jps.20221006.14

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  • @article{10.11648/j.jps.20221006.14,
      author = {Wang Jiaying and Zhao Lei and Li Wen and Huang Suwen and Zhang Jihong},
      title = {Visual Detection of Rf2 Event in Transgenic Rapeseed (Brassica napus L.) Using Loop-Mediated Isothermal Amplification},
      journal = {Journal of Plant Sciences},
      volume = {10},
      number = {6},
      pages = {222-226},
      doi = {10.11648/j.jps.20221006.14},
      url = {https://doi.org/10.11648/j.jps.20221006.14},
      eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.jps.20221006.14},
      abstract = {Specific methods have been developed in order to efficiently detect GMOs (genetically modified organisms) in both feed and food. Current approaches often rely on thermal cycling devices such as PCR amplifier, which makes it difficult for applications in the wild. Therefore a visual LAMP (Loop-Mediated Isothermal Amplification) method for rapid screening Rf2 event in transgenic rapeseed (Brassica napus L.) is established in this paper. For primer design, position 167-366 in the right border junction sequence of B. napus trangenic line Rf2 (accession number: EU090197.1) was chosen as the amplicon. Reaction mixture (at the volume of 25 μL) consisted of 12.5 μL 2×buffer mix, 1.28 μM FIP, 1.28 μM BIP, 0.16 μM F3, 0.16 μM B3, 480 U/mL Bst DNA polymerase, 2.0 μL DNA template and 5.0 μL ddH2O. Dye (SYBR Green I) was pre-added onto the tube lid. The reaction tube was incubated at 60°C for 60 min, followed by heating at 80°C for 10 min to end it. To mix dye with reaction mixture, the tube was centrifuged for 60 s at 8,000 r/min. Corresponding analysis results indicate that this LAMP assay is highly specific and sensitive (1.15×103 copies/μL). In one word, the visual LAMP method specific for Rf2 event in rapeseed, which turns out to be simple, time-effective, sensitive without relying on expensive instruments, is suitable for quick screening in ports.},
     year = {2022}
    }
    

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  • TY  - JOUR
    T1  - Visual Detection of Rf2 Event in Transgenic Rapeseed (Brassica napus L.) Using Loop-Mediated Isothermal Amplification
    AU  - Wang Jiaying
    AU  - Zhao Lei
    AU  - Li Wen
    AU  - Huang Suwen
    AU  - Zhang Jihong
    Y1  - 2022/11/29
    PY  - 2022
    N1  - https://doi.org/10.11648/j.jps.20221006.14
    DO  - 10.11648/j.jps.20221006.14
    T2  - Journal of Plant Sciences
    JF  - Journal of Plant Sciences
    JO  - Journal of Plant Sciences
    SP  - 222
    EP  - 226
    PB  - Science Publishing Group
    SN  - 2331-0731
    UR  - https://doi.org/10.11648/j.jps.20221006.14
    AB  - Specific methods have been developed in order to efficiently detect GMOs (genetically modified organisms) in both feed and food. Current approaches often rely on thermal cycling devices such as PCR amplifier, which makes it difficult for applications in the wild. Therefore a visual LAMP (Loop-Mediated Isothermal Amplification) method for rapid screening Rf2 event in transgenic rapeseed (Brassica napus L.) is established in this paper. For primer design, position 167-366 in the right border junction sequence of B. napus trangenic line Rf2 (accession number: EU090197.1) was chosen as the amplicon. Reaction mixture (at the volume of 25 μL) consisted of 12.5 μL 2×buffer mix, 1.28 μM FIP, 1.28 μM BIP, 0.16 μM F3, 0.16 μM B3, 480 U/mL Bst DNA polymerase, 2.0 μL DNA template and 5.0 μL ddH2O. Dye (SYBR Green I) was pre-added onto the tube lid. The reaction tube was incubated at 60°C for 60 min, followed by heating at 80°C for 10 min to end it. To mix dye with reaction mixture, the tube was centrifuged for 60 s at 8,000 r/min. Corresponding analysis results indicate that this LAMP assay is highly specific and sensitive (1.15×103 copies/μL). In one word, the visual LAMP method specific for Rf2 event in rapeseed, which turns out to be simple, time-effective, sensitive without relying on expensive instruments, is suitable for quick screening in ports.
    VL  - 10
    IS  - 6
    ER  - 

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Author Information
  • Technical Center, Ningbo Customs, Ningbo, China

  • Ningbo Institute of Inspection and Quarantine Science and Technology, Ningbo, China

  • Department of Horticultural Technology, Ningbo City College of Vocational Technology, Ningbo, China

  • Technical Center, Ningbo Customs, Ningbo, China

  • Technical Center, Ningbo Customs, Ningbo, China

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